The mammalian acute phase response is the first line of systemic defence elicited by stimuli such as infection, trauma, myocardial infarction, neoplasms, and surgery. It is initiated and maintained by a large number of pro-inflammatory mediators including cytokines, glucocorticosteroids and anaphylatoxins and involves a wide range of complex physiological changes including elevated circulating concentrations of hepatically synthesised acute phase reactants (APRs). In man, this latter class includes the "major" APRs, serum amyloid A (SAA) and C-reactive protein (CRP) (reviewed by Steel, D. M. and Whitehead, A. S. (1994) Immunol. Today (England) 15, 81).
The human SAA gene family is comprised of four known genes that have been localised to the short arm of chromosome 11p15.1. The SAA1 and SAA2 genes specify the two acute phase SAA proteins A-SAA1 and A-SAA2 respectively which are both 104 amino acid, 12.5 kDa proteins that share 93% amino acid sequence identity. A number of allelic forms have been identified by amino acid sequence analysis of A-SAA isolated from plasma. The A-SAA1 protein has three allelic forms whereas the A-SAA2 protein has two. A third gene SAA3 which shows 71% nucleotide identity with SAA1 and SAA2 is a pseudogene. Constitutive SAA (C-SAA) is the third expressed SAA family member and is the product of the SAA4 gene. C-SAA levels characteristically do not increase as a result of inflammation and exist in serum at concentrations between 80-140 mg/L (Yamada, T. et al. (1994) Int. J. Exp. Clin. Invest. 1, 114). C-SAA differs from A-SAA with respect to peptide length, being eight amino acids longer, and shares only 55% identity with A-SAAs. Additionally, C-SAA may be post-translationally modified by glycosylation at a single site. In common with the A-SAAs, C-SAA rapidly associates with high density lipoprotein (HDL3) when released into the circulation.
Circulating concentrations of A-SAA can increase up to 1000 mg/L within 24-48 hours of an acute stimulus (Marhaug, G. (1983); Scand. J. Immunol. 18, 329) indicating an important protective role for these proteins; however, no definitive function has been demonstrated for the A-SAA proteins. Recent studies variously suggest that A-SAA has chemoattractant activity, may play a role in lipid metabolism and immunosuppression and may inhibit the oxidative burst in neutrophils.
During chronic inflammation A-SAA levels remain significantly elevated reflecting the continued persistence of underlying pathological inflammatory processes that can contribute to long term tissue damage. An occasional consequence of chronic inflammation is reactive secondary amyloidosis, a progressive fatal condition in which amyloid A protein (AA), a cleavage product of A-SAA, is the major component of insoluble fibrous deposits that accumulate in major organs. The sustained elevation of A-SAA in chronic inflammatory conditions suggests that A-SAA is an important indicator of disease status. However, the measurement of A-SAA concentration has not been used for routine clinical diagnosis and clinical management, due in part to the difficulty in raising specific antisera against human A-SAA (Pepys, M. B. et al. (1984); British Medical Journal 288, 859).
Several methods, however, have been reported for the measurement of SAA levels: these include (i) radioimmunoassays and single radial immunodiffusion procedures (Chambers, R. E. and Whicher, J. T. (1983); J. Immunol. Methods 59, 95; Marhaug, G. (1983) supra; Taktak, Y. S. and Lee, M. A. (1991); J. Immunol. Methods 136, 11); (ii) ELISA based assays (Zuckerman, S. H. and Suprenant, Y. M.(1986); J. Immunol. Methods 92, 3743; Dubois, D. Y. and Malmendier, C. L. (1988); J. Immunol. Methods 112, 71-75; Sipe, J. D. et al. (1989); J. Immunol. Methods 125, 125-135; Yamada, T. et al. (1989); Clin. Chim. Acta 179, 169-176; Tino-Casl, M. and Grubb, A. (1993); Arm. Clin. Biochem 30, 278-286); (iii) nephelometric methods (Vermeer, H. et al. (1990); Clin. Chem 36, 1192; Yamada, T. et al. (1993); Ann. Clin. Biochem. 30, 72-76); (iv) an electrophoretic procedure (Godenir, N. L. et al. (1985); J. Immunol. Methods 83, 217); (v) an immunochemiluminescence procedure (Hachem, H. et al. (1991); Clin. Biochem 24, 143-147); (vi) an automated method based on a monoclonal-polyclonal antibody solid phase enzymeimmunoassay (Wilkins, J. W. et al. (1994); Clin. Chem 40(7), 1284-1290); and (vii) time-resolved fluorometric immunoassay (Malle, E., et al. (1995); J. Immunol. Methods 182, 131). As SAA in serum exists as one of the apolipoproteins associated with HDL3 particles many of these methods require denaturation of the serum samples (in an effort to eliminate the masking effect previously observed to be a problem in the accurate quantification of SAA) prior to carrying out the assay. Many assays previously reported have either measured total SAA or have been based on anti-sera raised against total SAA and have not been documented as being able to distinguish between the A-SAA and C-SAA proteins. Furthermore, many of these assays require an overnight incubation.
Problems have been encountered obtaining a soluble purified native A-SAA: purification of A-SAA protein from large volumes of blood is characterised by poor yields (Godenir, N. L. et al. (1985) supra), limited solubility (Bausserman, L. L. et al. (1983); J. Biol. Chem. 258, 10681) and the heterogeneous nature of the A-SAA recovered. In addition, A-SAA purified from serum may contain trace amounts of other serum components thereby potentially compromising studies of A-SAA function that involve sensitive bioassays.
There is a need for a method which provides a sensitive and reliable measure of A-SAA and inflammatory status which can be used for the diagnosis and clinical management of both acute and chronic inflammatory conditions.
WO 91/05874 is mainly concerned with total plasma protein immobilisation onto solid phases (e.g., polyvinyl chloride microtitre plates) and subsequent detection of said plasma proteins (e.g., SAA) using relevant antisera. The authors use inorganic salts and elevated temperatures as a means of promoting plasma protein adherence to the solid support. Reference is made to antigen capture ELISAs following specimen delipidation but this technique was found not to accurately and reproducibly facilitate detection of Serum Amyloid A. It is stated that antigen capture ELISAs do not provide the sensitivity required for clinically relevant measurements of SAA.
JP-A-63 0 44 895 refers to the generation of a monoclonal antibody, using a synthetic peptide, to a specific region to Serum Amyloid A protein which is proposed to have a potential utility in the diagnosis of secondary amyloidosis.
Biochemistry (1972), 11, 2934-2938 describes the primary sequence of a primate (Macaca mulatta) amyloid A protein and is concerned with the putative role of amyloid A in amyloidosis. No reference is made to the generation of antisera against the protein. While a fairly high degree of sequence homology is observed with the human activatable form of Serum Amyloid A it is unclear as to whether both proteins perform the same in vivo role in the two different species (Homo sapiens and Macaca mulatta).
Chemical Abstracts Vol. 125, No. 15 relates to the detection of Serum Amyloid A using antisera raised again the protein purified from human serum. The protein fraction purified from serum cannot be guaranteed to be free of the constitutive form and therefore cross-reactivity in any resultant ELISA test is a distinct possibility.